Numerous tropical infectious diseases such as malaria, encephalitis, sleeping sickness and viral fevers are spread by insect vectors. This laboratory has been examining the biochemical mechanism and enzymology associated with the construction of insect cuticle with a view to develop novel pest, and hence disease control measures. The specific aims are: a) to unravel the chemical and biological mechanisms causing the hardening of cuticle that protects the insects; b) to examine the enzymological aspects associated with sclerotization and melanization pathways that are responsible for cuticular hardening, defense reaction and wound healing; and c) to unravel the molecular biological aspects of phenoloxidase, a key enzyme associated with cuticle construction. To achieve these goals, model sclerotization studies with stable quinone methides and natural sclerotizing agents will be conducted. The mechanism of adduct formation between chitin (or its smaller oligomeric structures) and quinone methides and quinone methide imine amides will be investigated. The reactivities of quinone methide imine amide generated from 1,2-dehydro-N-acyldopamines with proteins and small molecules will be examined to understand the cross-linking mechanism occurring in vivo. The origin of ketocatechol in beta-sclerotized cuticle will be examined with the use of labeled dopamine derivatives. Model sclerotization studies will be continued with insect proteins and enzymatically generated sclerotizing agents. The cross-links formed in insect cuticle under natural as well as artificial conditions will be determined. Biochemical aspects of new proteins associated with cuticular hardening and melanization will be examined with special emphasis on multicomponent protein(s) and phenoloxidase inhibitor. The antibodies to the multienzyme complex will be raised and used to find out the differences between this enzyme and the monomeric proteins found in hemolymph. The possible occurrence of a multifunctional quinone isomerase/dopachrome tautomerase will be examined. The cDNA for cuticular phenoloxidase will be isolated for comparison of its sequence with the recently determined hemolymph phenoloxidase sequence. Expression of phenoloxidase gene in vectors would provide large amounts of the enzyme necessary for determining the structure-function relationship of phenoloxidase.